Iron chelating siderophores and several peptide antibiotics are assembled by nonribosomal peptide synthase complexes via a thioltemplate mechanism. The siderophore Yersiniabactin, a key virulence factor in Yersinia infections, has both an aryl-N-Cap initiating biosynthesis and five membered sulfur heterocycles (thiazolines, thiazolidines resulting from cyclization of cysteines) that coordinate iron. The research proposed here involves determination of the genes responsible for yersiniabactin biosynthesis and characterization of the first enzymatic steps that involve activation of salicylic acid, amide bond formation to cysteine residues and cyclization to the thiazoline. We propose to purify YbtE and domains for the 200KD high molecular weight protein 2 of Y. pestis and analyze the anticipated enzymatic activity, including posttranslational phosphopantetheinylation (Ppant), salicyl-S-Ppant enzyme loading, salicyl-cys-S-enzyme formation, and cyclization to a salicyl-thiazoline-S-enzyme intermediate. These studies should decipher the logic of enzymatic assembly not only of virulence-conferring siderophores yersiniabactin and anguibactin but also for peptide antibiotics such as actinomycin, pristinamycin, and the antitumor agent bleomycin which use aryl N-cap imitation and/or thiazoline, oxazline- forming steps. The research will utilize the enzymology expertise in the PI s group and the yersiniabacteria genetic, microbiology and pathogenesis expertise in the co-investigator s group to decipher the molecular mechanisms for virulence determining siderophore biogenesis.